Treat the “Untreatable” by a Photothermal Agent: Triggering Heat and Immunological Responses for Rabies Virus Inactivation

Abstract Rabies is a fatal neurological zoonotic disease caused by the rabies virus (RABV), and the approved post‐exposure prophylaxis (PEP) procedure remains unavailable in areas with inadequate medical systems. Although strategies have been proposed for PEP and postinfection treatment (PIT), because of the complexity of the treatment procedures and the limited curative outcome, developing an effective treatment strategy remains a holy grail in rabies research. Herein, a facile approach is proposed involving photothermal therapy (PTT) and photothermally triggered immunological effects to realize effective PEP and PIT simultaneously. The designed photothermal agent (N+TT‐mCB nanoparticles) featured positively charged functional groups and high photo‐to‐heat efficiency, which are favorable for virus targeting and inactivation. The level of the virus at the site of infection in mice is significantly decreased upon treatment with orthotopic PTT, and the transfer of the virus to the brain is significantly inhibited. Furthermore, the survival ratio of the mice three days postinfection is increased by intracranial injection of N+TT‐mCB and laser irradiation. Overall, this work provides a platform for the effective treatment of RABV and opens a new avenue for future antiviral studies.


Materials
All the chemicals and reagents were purchased from chemical sources, and the solvents for chemical reactions were distilled before use. Benzo [1,2-c:4,5-c′] bis ([1,2,5] thiadiazole) was purchased from Derthon Optoelectronic Materials Science Technology Co LTD. All air-and moisture-sensitive reactions were carried out in flame-dried glassware under a nitrogen atmosphere.
Measurements 1 H and 13 C NMR spectra were recorded at room temperature on a Unity-400 NMR spectrometer using CDCl 3 or DMSO as solvents and tetramethylsilane as a reference. Mass spectrometry (MS) was conducted with a GCT premier CAB048 mass spectrometer in matrixassisted laser desorption-ionization time-of-flight (MALDI-TOF) mode. Dynamic light scattering (DLS) was measured on a 90 plus particle size analyser. Transmission electron microscopy (TEM) images were acquired on a JEM-2010F transmission electron microscope with an accelerating voltage of 200 kV. Density functional theory (DFT) calculations were carried out at the B3LYP/6G(d) level of theory by applying the Gaussian 09 package.
The mixture was stirred in a fume hood under N 2 bubbling overnight to remove the THF. N + TT-mCB NPs were subjected to ultrafiltration (molecular weight cut-off of 100 kDa) at 3000 × g for 30 min.

Cell viability assay
The cytotoxicity of N + TT-mCB NPs in BSR cells was determined by CCK-8 assay, which was performed according to the manufacturer's instructions (Beyotime, China). Briefly, cells were seeded at 3×10 4 per well into 96-well plates and cultured overnight. When the cells grew to 60-70%, the medium was changed to DMEM supplemented with different concentrations of N + TT-mCB NPs to continue the incubation for 24 h. Then, 10 μL CCK8 was added to each well and incubated at 37 °C for 1 h. The absorbance at 450 nm was detected with a microplate reader. The cell survival rate was calculated with the absorbance value.

RNA isolation and quantitative real-time PCR (qPCR)
The mouse brains and muscles were homogenized separately and centrifuged at 4 ℃, 8000 r.p.m. for 5 min. RNA was isolated from the cell pellet using TRIzol reagent (0.1 g/ml) according to the manufacturer's instructions (TIANGEN, China) and reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit for qPCR (TAKARA, Japan). The standard curve was set with the positive plasmid pcDNA3.1-RABV N. The following primers were

H&E staining and IHC
Organizations (muscle, brain) were placed in 10% formaldehyde solution and fixed for at least 24 h. The tissue was dehydrated in gradient alcohol, cleared in xylene, paraffin, and embedded. Then, the slices were dewaxed with xylene, rehydrated with 100%, 90%, 80% anhydrous ethanol in turn, put into haematoxylin staining solution for 5 min, rinsed with tap water, dehydrated with 80% alcohol and stained in eosin dye solution for 3 min; the sections were put into 80%, 90%, and 100% anhydrous ethanol I and II and xylene I and II in turn for 3 min each time, dehydrated and cleared in turn. The slices were removed, and the tissue surface was sealed with drops of neutral gum resin.
The mouse muscles and brains were fixed in 10% formaldehyde solution. The fixed brains were embedded in paraffin wax and cut into 4-5 μm-thick sections. After antigen retrieval in citric acid buffer, block endogenous peroxidase in 30% hydrogen peroxide for 20 min in IHC.
The mouse brain sections were incubated with mouse anti-IL-6 antibody to detect inflammation.  Synthetic route of 8. Add nBuLi (0.7 mL, 1.7 mmol, 2.4 M in hexane) dropwise to a solution of 7 (1 g, 1.7 mmol) in THF (30 mL) at -78 °C. The reaction mixture was stirred for 1 h at -78 °C. Then, trimethyltin chloride (1.7 mL, 1.7 mmol, 1.0 M in THF) was added to the reaction at one portion. After stirring the mixture for 12 h at room temperature, KF solution was added to quench the reaction. The mixture was extracted with hexane three times, and the combined organic phase was dried with Na 2 SO 4 . After removing the solvent, the product was used directly without further purification.
Synthetic route of NTT-mCB. To a solution of compounds 4 (161 mg, 0.15 mmol) and 8 (261 mg, 0.4 mmol) in toluene (10 mL) was added Pd (PPh 3 ) 4 (4 mg). The mixture was stirred for 12 h at 100 °C. After cooling to room temperature, the mixture was poured into water and extracted with DCM. The organic layer was washed with saturated KF and brine before being    To a solution of compound NTT-mCB (100 mg) in acetone (5 mL) was added MeI (1 mL).
The mixture was refluxed overnight. After cooling to room temperature, the mixture was subjected to rotary evaporation. The crude product was purified by DCM washing several times.